The proton-coupled di- and tri-peptide transporter isoform PepT1 has an exceptionally wide substrate range, leading to interest in understanding the substrate-binding site (see Meredith & Boyd, 2000 for a review). Here we report the results of site-directed mutagenesis experiments to test whether particular highly conserved residues play a predictable role in PepT1 function, with the aim of further refining our substrate template model (Bailey et al. 2000).
Selected rabbit PepT1 residues were mutated using a PCR-based protocol (Quikchange, Stratagene) and confirmed by DNA sequencing. Uptake experiments into PepT1-expressing Xenopus laevis oocytes were performed as previously described (Meredith et al. 2000). Data are means ± S.E.M.; n = 5 oocytes.
His121 has been proposed as playing a role in the binding of the side chain of the second residue in a dipeptide, with the protonated form (HisH+) donating a proton to the COO– of acidic residues (Chen et al. 2000). The H121A, H121N and H121F PepT1 mutations were virtually unaffected, as might be predicted for uptake of a neutral substrate (Fig, 1). Arg282 has been predicted from computer modelling to be involved in regulating translocation of substrates through PepT1, although the Vmax of R282A-hPepT1 was only reduced 15 % (Bolger et al. 1998). Indeed, as can be seen in Fig. 1, even after swapping the wild-type positively charged residue for a negative glutamate, R282E-PepT1 could still perform transport. Finally, Glu594 has been proposed as the binding site for the amino terminus NH3+ group of substrates (Meredith et al. 2000). Conservative substitution of this residue for negatively charged Asp (E594D, Fig. 1) abolished uptake, suggesting that simply the presence of a negatively charged amino acid residue at this point in the sequence is insufficient for functional protein.
These preliminary results confirm and extend existing studies implicating His121 and Glu594 in the functioning of the PepT1 transport protein. The role of Arg282 is currently less clear. Further studies will be required to elucidate the full roles of these residues in PepT1 in substrate binding and translocation.
This work is generously funded by the Wellcome Trust.